To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis thaliana, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.
Because there is no morphogenetic movement of plant cells, control of the three-dimensional structure of organs is only through regulation of cell division and cell expansion. Distinct from most other eukaryotes, after division, plant cells dramatically increase their size achieving volumes that can be hundreds of times their original size (Cosgrove, D. J., Annu. Rev. Cell. Dev. Biol. 13: 171–201, 1997). For plant organs to attain their final morphology and function properly, constituent cells must tightly regulate the way in which they expand. The orientation and extent of an individual cell's expansion are key parameters in determining its size and shape, yet little is known about the molecular mechanisms that regulate either aspect of cell expansion.
Although cell expansion is driven by cell turgor, all evidence indicates that neither water flow nor solute influx to maintain turgor is the primary determinant of the extent or direction of cell expansion (Pritchard, J., New Phytol. 127: 3–26, 1994); rather, the plant cell wall is believed to be the regulator of both. The plant cell wall comprises an array of para-crystalline cellulose microfibrils, which are associated with cross-linking glycans (e.g, hemicellulose) and embedded in a matrix of pectin and small amounts of protein (McCann, M. C., et al., “Architecture of the primary cell wall.” in The cytoskeletal basis of plant growth and form (ed, C. W. Lloyd), pp. 109–129. Academic Press, London, 1991; Carpita, N. C., et al., Plant J. 3, 1–30, 1993). The polysaccharides of the growing plant cell wall are mostly long-chained polymers that form a cohesive network through non-covalent lateral associations and physical entanglements (Cosgrove, D. J., Ann. Rev. Plant Physiol. Plant Mol. Biol. 50: 391–417, 1999). The cell wall's ability to withstand enormous osmotic pressure while readjusting the arrangement of these constituent polymers appears to be critical to the expansion process.
Regulation of the direction in which a cell expands involves oriented control of cell wall extension as well as polarized deposition of new wall materials (Carpita, N. C., et al., Plant J. 3, 1–30, 1993). Biophysical considerations indicate that there must be a component in the expanding walls that resists the osmotic pressure, thereby channeling the direction of cell elongation. Most evidence points to cellulose microfibrils as the primary load-bearing component of the expanding cell wall performing this function (Pritchard, J., New Phytol. 127: 3–26, 1994). In many cell types, cellulose microfibrils have been shown to be oriented perpendicular to the primary direction of expansion, analogous to hoops around a barrel (Green, P. B., Ann. Rev. Plant. Physiol. 51–82, 1980; Giddings, T. H. J., et al., “Microtubule-mediated control of microfibril deposition: A re-examination of the hypothesis” in The cytoskeletal basis of plant growth and form (ed, C. W. Lloyd), pp. 85–99. Academic, London, 1991). Therefore, to regulate the orientation of cell expansion, the cell must be able to control the deposition and spatial organization of cellulose microfibrils as well as rearrange bonds to allow the wall to yield to or resist the osmotic pressure. Unlike pectins and cross-linking glycans, which are made in the cytoplasm and transported out to the wall via the Golgi apparatus (Gibeaut, D. M., et al., FASEB J. 8: 904–915, 1994), cellulose microfibrils are synthesized at the cell membrane-cell wall interface (Delmer, D. P., Plant Mol. Biol. 50: 245–276, 1999). The cellulose microfibrils that are spooled around plant cells are generated by multimeric protein complexes in the plasma membrane commonly referred to as the “terminal complexes” or “particle rosettes”. About three dozen individual polymer chains of (1–4)-β-D-glucans are synthesized and subsequently crystallized into a microfibril (Delmer, D. P., Plant Mol. Biol. 50: 245–276, 1999). The process of microfibril crystallization may be facilitated by a subunit of the rosette complex (Delmer, D. P., et al., Plant Cell 7: 987–1000, 1995). Recently, genes have been identified that are involved either in the synthesis of cellulose (Arioli, T., et al., Science 279: 717–720, 1998; Pear, J. R., et al., Proc. Natl. Acad. Sci. USA. 93: 12637–12642, 1996; Turner, S. R., et al., Plant Cell 9: 689–701, 1997; Taylor, N. G., et al., Plant Cell 11: 769–780, 1999; Fagard, M., et al., The Plant Cell, 12, 2000 (in press)) or of non-cellulosic polysaccharide components at the Golgi apparatus or involved in their secretion to the wall (Lukowitz, et al., Cell 84, 61–71, 1996; Bonin, C. P., et al., Proc. Natl. Acad. Sci. USA 94: 2085–2090, 1997; Nicol, F., et al., EMBO J. 17: 5563–5576, 1998; Edwards, M. E., et al., Plant J. 19, 691–697, 1999; Perrin, R. M., et al., Science 284: 1976–1979, 1999; Gibeaut, D. M., Plant Physiol. Biochem. 38: 69–80, 2000). Genes and their products have also been identified that function in rearranging bonds in the cell wall to allow for extensibility (Cosgrove, D. J., Ann. Rev. Plant Physiol. Plant Mol. Biol. 50: 391–417, 1999). In particular, expansins are a family of proteins involved in the disruption of the non-covalent bonds between cellulose microfibrils and cross-linking glycans, causing rapid induction of wall extension (McQueen-Mason, S., et al., Proc. Natl. Acad. Sci. USA 91: 6574–6578, 1994). However, regulation of the orientation or extent of expansion is still poorly understood at the molecular level. To further understand the molecular mechanisms involved in cell expansion we have cloned the COBRA (COB) gene and determined its pattern of expression in roots. Initially cobra, a member of the conditional root expansion (CORE) class of mutants, was isolated in a screen for Arabidopsis seedlings with abnormally expanded roots (Benfey, P. N., et al., Development 119: 57–70, 1993). The phenotype of all CORE mutants is conditional on the root growing in the presence of high concentrations of sucrose or other conditions that stimulate rapid root growth (Hauser, M. T., et al., Development 121: 1237–1252, 1995). Root cells in cob appear to be expanded more in the radial than the longitudinal orientation while maintaining cell volume, indicating a role for COBRA in regulating the orientation of cell expansion. Here we report that COB encodes a putative GPI-anchored protein that is localized primarily in the plasma membrane of the longitudinal sides of root cells, and plays a role in determining the orientation of cell expansion.